Plan apochromat 100x na 1.4 oil

To measure the association of SNAP25 constructs with the plasma membrane on plasma membrane sheets, a Zeiss Axio Observer D1 epifluorescence microscope equipped with a Plan-Apochromat 100x/NA 1.4 oil immersion objective and a 12 bit CCD camera (1376 × 1040 pixel) was used, yielding a pixel size of 64.5 nm x 64.5 nm. Freshly prepared membrane sheets were imaged in sonication buffer supplemented with TMA-DPH (1-(4-tri-methyl-ammonium-phenyl)−6-phenyl-1,3,5-hexatriene-p-toluenesulfonate; Thermo Fisher Scientific), up to ≈ 35 min after membrane-sheet generation. TMA-DPH staining was applied for visualization of the shape and integrity of the membrane sheets. Pictures were taken using filter sets F11-000 (AHF Analysentechnik, Tübingen, Germany) for TMA-DPH (blue channel) and F36-525 (AHF Analysentechnik, Tübingen, Germany) for GFP (green channel). From individual membrane sheets, the fluorescence intensity was measured in 30 pixel x 30 pixel ROIs and background subtracted. For each experiment and condition, the values of 21–96 membrane sheets were averaged.

Confocal immunofluorescence microscopy: The images of antibody-stained polytene chromosomes and imaginal discs were captured using an upright confocal microscope (Carl Zeiss LSM 710) using following objective lenses: Plan Apochromat 25x/NA 0.8 Oil, Plan Apochromat 40x/NA 1.4 Oil, Plan Apochromat 100x/NA 1.4 Oil (all Carl Zeiss), at 23 °C. Secondary antibodies conjugated to two fluorochromes were used, Alexa Fluor 488 (Goat anti-Rabbit) and Alexa Fluor 594 (Goat anti-mouse). The images were acquired using Zen 2010 acquisition software (Carl Zeiss) and further processed using ImageJ (NIH). Only the brightness and contrast were altered for entire image without changing the grey values.
Scanning electron micrograph imaging: One day prior to imaging, flies were collected and stored at 4 °C in a 1.5 mL tube. On the day of imaging, flies were kept on ice. Flies were mounted on the imaging stubs under a dissection microscope using a needle and enough pressure was applied so as to stick the flies to the double-sided tape placed on the stub. Imaging was performed at 10,000 psi. Images were acquired on Scanning electron microscope FVO-LS10 (Zeiss) at multiple magnifications, 95 ×, 120 ×, 400 ×, and 750 ×. Using ImageJ, only, the brightness and contrast of the images were altered without changing the grey values.